Under almost all conditions, using any method, the levels of gene transfer to any cell are low because many barriers exist for the efficient delivery of genes to cells. Taken one step further, gene transfer to tissues within living animals is even worse, at least in part since cells in all tissues are constantly exposed to mechanical stresses such as shear, compression, and stretch. Thus, we must elucidate the pathways and molecular mechanisms of gene delivery under static conditions and those of mechanical strain if we are to increase the efficacy of gene therapy. Mechanical stretch induces numerous biological responses in cells that are directly related to the process of gene delivery. Exogenous DNA must enter the cell, cross the cytoplasm, enter the nucleus, and be expressed for gene therapy to be successful. We have shown that gene delivery and expression in multiple cell types exposed to equibiaxial stretch is 10-fold more efficient than in cells grown under static conditions. We have also shown that this cyclic stretch reorganizes the cytoskeleton and increases the numbers of stable, acetylated microtubules by a mechanism mediated by inhibition of the cytoplasmic histone deacetylase HDAC6, whose main target is 1-tubulin. It has been shown that microtubule acetylation causes the recruitment of dynein motors and increases cytoplasmic trafficking of bound cargoes. We hypothesize that cyclic stretch modulates HDAC6 activity resulting in increased levels of acetylated microtubules and increased cytoplasmic trafficking of plasmid DNA-protein complexes toward the nucleus for enhanced gene expression. Although not a physiologically "normal" process, the interactions of plasmids with the host cell are vital to methods that scientists use every day and form the basis of gene therapy. The experiments in this application will elucidate the mechanisms of stretch-enhanced gene delivery in cultured cells and living animals, with a focus on the alveolar epithelium, a tissue that continuously undergoes cyclic stretch. Finally, we have developed an electroporation method for high-level, safe, non-viral gene transfer to the lung and have used this approach to treat lung injury in experimental animal models. We will now utilize this approach to explore the mechanisms of in vivo gene transfer and develop treatment approaches for the injured lung. The specific aims are to (1) determine the role of HDAC6 and acetylated microtubules in cyclic stretch-enhanced gene transfer in cells, (2) identify the components of the DNA-protein complex that facilitate movement of plasmids through the cytoplasm in stretched and unstretched cells, and (3) determine whether mechanical ventilation increases gene transfer to the alveolar epithelium in the mouse lung in vivo through HDAC6. PUBLIC HEALTH RELEVANCE: Gene therapy is an exciting and potentially very useful approach to treat a number of diseases at the molecular level. Unfortunately, many barriers for gene delivery to cells and animals exist that must be characterized before they can be overcome, leading to greater levels of gene transfer and gene therapy. Although most work on gene transfer has been studied in cells growing undisturbed in dishes, most cells in the body, especially those in the lung, our target organ of interest, are constantly undergoing various forms of mechanical strain including cyclic stretch. We will determine the molecular mechanisms by which the cells respond to cyclic stretch to rearrange their cytoskeleton and increase intracellular DNA movement and gene therapy in isolated cells and small animal models.